Parkinson's Disease
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of the nigrostriatal pathway. Although the cause of Parkinson's disease is not known, it is associated with the progressive death of dopaminergic (tyrosine hydroxylase (TH) positive) mesencephalic neurons, inducing motor impairment. The characteristic symptoms of Parkinson's disease appear when up to 70% of TH-positive nigrostriatal neurons have degenerated.
There is currently no satisfactory cure for Parkinson's disease. Symptomatic treatment of the disease-associated motor impairments involves oral administration of dihydroxyphenylalanine (L-DOPA). L-DOPA is transported across the blood-brain barrier and converted to dopamine, partly by residual dopaminergic neurons, leading to a substantial improvement of motor function. However, after a few years, the degeneration of dopaminergic neurons progresses, the effects of L-DOPA are reduced and side-effects reappear. Better therapy for Parkinson's disease is therefore necessary.
An alternative strategy for therapy is neural grafting, which is based on the idea that dopamine supplied from cells implanted into the striatum can substitute for lost nigrostriatal cells. Clinical trials have shown that mesencephalic TH positive neurons obtained from human embryo cadavers (aborted foetuses) can survive and function in the brains of patients with Parkinson's disease. However, functional recovery has only been partial, and the efficacy and reproducibility of the procedure is limited. Also, there are ethical, practical and safety issues associated with using tissue derived from aborted human foetuses. Moreover, the large amounts of tissue required to produce a therapeutic effect is likely to prove to be prohibitive. Some attempts have been made to use TH positive neurons from other species (in order to circumvent some of the ethical and practical problems). However, xenotransplantation requires immunosuppressive treatment and is also controversial due to, for example, the possible risk of cross-species transfer of infectious agents. Another disadvantage is that, in current grafting protocols, no more than 5–20% of the expected numbers of grafted TH positive neurons survive. In order to develop a practicable and effective transplantation protocol, an alternative source of TH positive neurons is required.
A further alternative strategy for therapy is gene therapy. It has been suggested that gene therapy could be used in Parkinson's disease in two ways: to replace dopamine in the affected striatum by introducing the enzymes responsible for L-DOPA or dopamine synthesis (for example, tyrosine hydroxylase); and to introduce potential neuroprotective molecules that may either prevent the TH-positive neurons from dying or stimulate regeneration and functional recovery in the damaged nigrostriatal system (Dunnet S. B. and Björklund A (1999) Nature 399 A32–A39).
In vivo, dopamine is synthesised from tyrosine by two enzymes, tyrosine hydroxylase (TH) and aromatic amino acid DOPA-decarboxylase (AADC). Parkinson's disease has been shown to be responsive to treatments that facilitate dopaminergic transmission in caudate-putamen. In experimental animals, genetically modified cells that express tyrosine hydroxylase, and thereby synthesise L-DOPA, induce behavioural recovery in rodent models of PD (Wolff et al. (1989) PNAS (USA) 86:9011–14; Freed et al (1990) Arch. Neurol. 47:505–12; Jiao et al. (1993) Nature 262:4505).
Functional activity of tyrosine hydroxylase depends on the availability of its cofactor tetrahydrobiopterin (BH4). The level of cofactor may be insufficient in the denervated striatum, and so it is thought that GTP cyclohydrolase I, the enzyme that catalyses the rate limiting step on the pathway of BH4-synthesis, may also need to be transduced to obtain sufficient levels of L-DOPA production in vivo (Bencsics et al (1996) J. Neurosci 16:4449–4456; Leff et al (1998) Exp. Neurol. 151:249–264).
Although in vivo and ex vivo gene therapy strategies for the treatment of Parkinson's disease have already been proposed (Dunnet and Bjorklund (1999) as above; Raymon et al (1997) Exp. Neurol. 144:82–91; Kang (1998) Mov. Dis. 13: 59–72) significant progress in this technology has been hampered by the limited efficiency of gene transfer and expression in the target cells. One problem in this regard is that the target cells are usually non-dividing cells (i.e. neurones) which are notoriously recalcitrant to transduction.
Expression of More than One Protein
WO 98/18934 relates to a a polynucleotide sequence for use in gene therapy, which polynucleotide sequence comprises two or more therapeutic genes operably linked to a promoter, and encodes a fusion protein product of the therapeutic genes. This provides a way of expressing two therapeutic genes from a single “chimeric gene”. In a preferred embodiment, the polynucleotide sequence is capable of encoding a fusion protein comprising tyrosine hydroxylase and DOPA decarboxylase in either TH-DD or DD-TH order, linked by a flexible linker.
As discussed in WO/18924, amongst gene transfer systems, retroviral vectors hold substantial promise for gene therapy. These systems can transfer genes efficiently and new vectors are emerging that are particularly useful for gene delivery to brain cells (Naldini et al., 1996 Science 272, 263). However, it is dear from the literature that retroviral vectors achieve the highest titres and most potent gene expression properties if they are kept genetically simple (PCT/GB96/01230; Bowtell et al., 1988 J. Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol. Cell. Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J. Biol. Chem 266, 8416; Hatzoglou et al., 1988 J. Biol. Chem 263, 17798; Li et al., 1992 Hum. Gen. Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol. Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197). This means using a single transcription unit within the vector genome and orchestrating appropriate gene expression from sequences either within the 5′ LTR or from an internal promoter using a self-inactivating LTR, or using the split-intron technology described in the WO99/15683.
According to WO 98/18934, if there is a need to express two proteins from a single retroviral vector it is preferable to express them as a fusion protein (encoded by a single nucleotide sequence) than to use an internal ribosome entry site (IRES) to initiate translation of the second coding sequence in a poly-cistronic message. This is because, according to WO 98/18934 the efficiency of an IRES is often low and tissue dependent making the strategy undesirable when one is seeking to maximise the efficiency of metabolic conversion of, for example, tyrosine through to dopamine.
When located between open reading frames in an RNA, an IRES allows translation of the downstream open reading frame by promoting entry of the ribosome at the IRES element followed by downstream initiation of translation. The use of IRES elements in retroviral vectors has been investigated (see, for example, WO 93/0314) but expression of the cDNA situated downstream of the IRES has often been found to be inefficient. This may be due to competition for ribosomes and other cellular factors. The efficiency of translation initiation would therefore be expected to decrease with increasing numbers of IRES elements.
Expression of Large Heterologous Genes
Although the concept of using viral vectors to deliver a heterologous gene to a recipient cell is well known (Verma and Somia (1997) Nature 389:239–242), it is widely accepted that there are limits on the size of the heterologous gene which can be successfully transduced (see, for example page 446, Chapter 9 of Coffin et al “Retroviruses” 1997 Cold Spring Harbour Laboratory Press). If incorporation of the heterologous gene and associated regulatory elements dramatically increases the size of the viral genome, then there is a significant risk that it will no longer be able to be successfully packaged, or at least that packaging efficiency will be significantly reduced.
Despite the apparent prejudice in the art, the present inventors have shown that lentiviral vectors expressing a bicistronic cassette (encoding TH and GTP-CH1) and even a tricistronic cassette (encoding TH, AADC and GTP-CH1) can yield expression of the appropriate enzymes in heterologous cells in culture and in vivo. Incorporation of the tricistronic cassette into the lentiviral vector causes an increase in the size of the RNA genome of approximately 10%–30% (over the wild-type RNA genome) but surprisingly, gene transfer efficiency is not markedly affected. Integration efficiencies are comparable and efficient gene transfer to neurons is demonstrated. Moreover, the inventors have shown that such vectors may be used to increase the levels of certain catecholamines in denervated tissue and therefore correct rodent and primate models of Parkinson's disease.